Aprotinin is a well characterized basic protein of 58 amino acids which acts as inhibitor of such proteinases as trypsin, chymotrypsin, plasmin and kallikrein. It has become a valuable drug, named Trasylol.RTM., for the treatment of various diseases such as, e.g., hyperfibrinolytic hemmorrhage and traumatic hemmorrhagic shock.
Recently it has been demonstrated that the replacement of the residue lysine in position 15 of the aprotinin molecule by other amino acids results in valuable proteinase inhibitors with a modified spectrum of inhibition compared to aprotinin (H. Tschesche et al. (1985), patent application DOS 33 39 693 of May 15, 1985. Depending on the amino acid introduced these modified inhibitors may, e.g., act as inhibitors of the elastases from pancreas and from leukocytes and/or plasma kallikrein. Although aprotinin variants may be obtained by semisynthetic conversion of aprotinin (H. Tschesche et al., DE-OS 33 39 693). The amounts obtainable are relatively small. In addition the methodology does not allow multiple replacements of amino acids in addition to the lysine residue in position 15.
It was, therefore, perceived that the application of recombinant DNA and associated technologies would be the most appropriate way of producing large quantities of aprotinin homologues with the desired specifity and efficacy of inhibition.
To those skilled in the art DNA coding for proteins of known amino acid sequence may be prepared using the genomic DNA-sequence or the cDNA-sequence which is complementary to the mRNA. Amino acid replacements may then be introduced by, e.g., site-directed mutagenesis. Another possibility of obtaining DNA coding for a protein of known primary structure is to choose codons according to the genetic code and to prepare a synthetic gene.
Methods for the expression of heterologous DNA in a recombinant microorganism and/or in eucaryotic cells are known.